1. Field of the Invention
The present invention relates to a fluorescence sensor that detects specific substances within samples by fluorometry. More specifically, the present invention relates to a fluorescence sensor that performs surface plasmon enhancement.
2. Description of the Related Art
Fluorometry is conventionally used in for biological measurements and the like, as an easy and highly sensitive measuring method. In fluorometry, a sample, which is considered to contain a detection target substance that emits fluorescence when excited by light having a specific wavelength, is irradiated with an excitation light beam of the aforementioned specific wavelength. The presence of the detection target substance can be confirmed by detecting the fluorescence due to the excitation. In the case that the detection target substance is not a fluorescent substance, a substance labeled by a fluorescent substance that specifically bonds with the detection target substance is caused to contact the sample. Thereafter, fluorescence is detected in the same manner as described above, thereby confirming the presence of the bond, that is, the detection target substance.
FIG. 3 is a diagram that illustrates the schematic structure of an example of a sensor that performs fluorometry employing the aforementioned labeled substance. The fluorescence sensor is that which detects antigens 2 included in a sample 1. Primary antibodies 4 that specifically bond with the antigens 2 are coated on a substrate 3. The sample 1 is caused to flow through sample holding portion 5 provided above the substrate 3. Next, secondary antibodies 6, which are labeled with a fluorescent substance 10 and also bond specifically with the antigens 2, are caused to flow through the sample holding portion 5. Thereafter, a light source 7 emits an excitation light beam 8 toward the surface of the substrate 3, and a photodetector 9 detects fluorescence. At this time, if a predetermined fluorescence is detected by the photodetector 9, bonds between the secondary antibodies 6 and the antigens 2, that is, the presence of the antigens 2 within the sample 1, is confirmed.
Note that in the above example, the presence of the secondary antibodies 6 is actually confirmed by detecting the fluorescence. However, if the secondary antibodies 6 do not bond with the antigens 2, then they will flow past the substrate 3, and will not be present thereon. Therefore, the presence of the antigens 2 is indirectly confirmed, by confirming the presence of the secondary antibodies 6.
With recent advances in the performance of photodetectors, such as cooled CCD's, fluorometry has become indispensable in biological research. In addition, fluorometry has come to be widely used in fields other than biology.
However, in conventional surface plasmon enhanced fluorescence sensors such as that illustrated in FIG. 3, the excitation light beam reflected or scattered at the interface between the substrate and the sample, and light scattered by impurities other than the detection target substance becomes noise. Therefore, even if the performance of photodetectors is improved, the S/N ratio in fluorescence detection does not.
As a solution to this problem, evanescent wave fluorometry, that is, fluorometry that employs evanescent waves, has been proposed in A. Kusumi et al., “This Much Can Be Learned From Bio Imaging”, pp. 104-113, Yodosha Press. An example of a fluorescence sensor that performs evanescent wave fluorometry is illustrated in FIG. 4. Note that in FIG. 4, elements which are the same as those illustrated in FIG. 3 are denoted with the same reference numerals, and detailed descriptions thereof will be omitted unless particularly necessary (the same applies to all of the following descriptions).
In this fluorescence sensor, a prism 13 (dielectric block) is employed instead of the substrate 3. The excitation light beam 8 emitted from the light source 7 is irradiated through the prism 13 such that conditions for total internal reflection at the interface between the prism 13 and the sample 1 are satisfied. In this configuration, the secondary antibodies 6 are excited by evanescent waves 11 that leak in the vicinity of the interface when the excitation light beam 8 is totally internally reflected thereat. Fluorescence detection is performed by the photodetector 9, which is provided at the side opposite the prism 13 from the sample 1 (the upper portion in FIG. 4).
In this fluorescence sensor the excitation light beam 8 is totally internally reflected toward the lower portion of the drawing, and fluorescence detection is performed from above. Therefore, detected excitation light components do not become a background for a fluorescence detection signal. In addition, the evanescent waves 11 only reach a region of several hundred nm from the interface. Therefore, scattering due to impurities M within the sample 1 can be virtually eliminated. For these reasons, evanescent fluorometry is being noticed as a method that enables fluorescent measurement of detection target substances in units of single molecules, while greatly reducing (light) noise compared to conventional fluorometry.
A surface plasmon enhanced fluorescence sensor, such as that illustrated in FIG. 5, is also known as a sensor capable of fluorescence measurement at even higher sensitivity. The surface plasmon enhanced fluorescence sensor is disclosed, for example, in Japanese Patent No. 3562912, and differs from the fluorescence sensor of FIG. 4 in that a metal film 20 is formed on the prism 13. That is, surface plasmon is generated within the metal film 20 when the excitation light beam 8 is irradiated thereon, and the electric field amplification effect provided thereby enhances the fluorescence. According to a simulation, the fluorescent intensity has been shown to be enhanced 1000 times.
However, in the aforementioned surface plasmon enhanced fluorescence sensor, if the fluorescent substance within the sample and the metal film are too close to each other, the energy excited within the fluorescent substance is transferred to the metal film before fluorescence is emitted. That is, a phenomenon in which fluorescence does not occur (so called metallic light loss) may occur.
Fang Yu et al. propose forming a SAM (Self Assembled Membrane) on the metal film, to separate the fluorescent substance within the sample from the metal film at least by a distance equal to the thickness of the SAM, in “Surface Plasmon Field-Enhanced Fluorescence Spectroscopy Studies of the Interaction between an Antibody and Its Surface-Coupled Antigen”, Analytical Chemistry, Vol. 75, pp. 2610-2617, 2003. Note that FIG. 5 illustrates the SAM, denoted by reference number 21. T. Liebermann et al. also discuss the dependency of the fluorescence intensity enhanced by surface plasmon on the distance from the metal film, related to metallic light loss, in “Surface-Plasmon Field-enhanced fluorescence Spectroscopy”, Colloids and Surfaces 171, pp. 115-130, 2000.
However, when fluorescence detection was performed using the aforementioned surface plasmon enhanced fluorescence sensor provided with the SAM, it was found that the sensitivity of fluorescence detection was not improved much, compared to a case in which the SAM is not provided.